PROJECT SUMMARY The goal of this project is to gain an in-depth mechanistic understanding about multi-heteromeric ion channels, N-methyl-D-aspartate receptors (NMDARs), regulated by different compounds, splice variants, and subtypes. These receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of excitatory synaptic transmission and play significant roles in basic brain functions, development, and neurological disorders such as seizures, strokes, depression, and schizophrenia, as well as Parkinson?s and Alzheimer?s diseases. NMDARs are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind the neurotransmitter, glutamate. Each subunit protein is composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their transmembrane ion channels upon binding of glycine and glutamate, whereas the GluN1-GluN3 NMDARs activate by glycine alone. We recently solved the first structure of the intact hetero-tetrameric rat GluN1-GluN2B NMDARs, initially by x-ray crystallography and later by cryo- electron microscopy. Despite recent advances, there are many fundamental questions remaining regarding the mechanism of activation, desensitization, inhibition by competitive antagonists, and functional diversity elicited by alternative splicing in GluN1 and different subunit combinations (GluN1-GluN2A-D and GluN3A-B). The structure of the GluN3 NMDARs is limited to that of the GluN3A LBD, thus, restricting our understanding about how this under-studied subtype form ion channels and mediate functions. Thus, the goal of the proposed project is to investigate patterns of compound bindings, protein conformations and subunit arrangements in various functional states and subtypes of NMDARs. To achieve these goals, we will conduct research aimed at: Aim 1 determining the mechanisms of activation, desensitization, and inhibition by agonists and antagonists; Aim 2 defining the underlying mechanism for altered pH-sensitivity and deactivation speeds by polyamines in a splice- variant-specific manner; and Aim 3 revealing the architecture of the GluN1-GluN3 NMDAR for the first time. These three aims will be achieved by obtaining the structural information of the GluN1-GluN2B NMDAR in the presence of agonists and antagonists, splice variants with and without spermine, and the GluN1-GluN3A NMDAR by a combination of x-ray crystallography and cryo-EM. The structure-based functional hypotheses will be tested by experiments involving electrophysiology. Successful completion of the proposed studies will provide in-depth information into the sophisticated function of NMDAR subtypes and splice variants mediated by compound bindings followed by rearrangement of multiple subunits and domains. These findings will support the development of therapeutic strategies used to treat the devastating neurological disorders and diseases mentioned above.